B-class MADS-box genes in trioecious papaya: two paleoAP3 paralogs, CpTM6-1 and CpTM6-2, and a PI ortholog CpPI

In the ABC model of flower development, B function organ-identity genes act in the second and third whorls of the flower to control petal and stamen identity. The trioecious papaya has male, female, and hermaphrodite flowers and is an ideal system for testing the B-class gene expression patterns in trioecious plants. We cloned papaya B-class genes, CpTM6-1, CpTM6-2, and CpPI, using MADS box gene specific degenerate primers followed by cDNA library screening and sequencing of positive clones. While phylogenetic analyses show that CpPI is the ortholog of the Arabidopsis gene PI, the CpTM6-1 and CpTM6-2 loci are representatives of the paralogous TM6 lineage that contain paleoAP3 motifs unlike the euAP3 gene observed in Arabidopsis. These two paralogs appeared to have originated from a tandem duplication occurred approximately 13.4 million year ago (mya) (bootstrap range 13.36 ± 2.42). In-situ hybridization and RT-PCR showed that the papaya B-class genes were highly expressed in young flowers across all floral organ primordia. As the flower organs developed, all three B-class genes were highly expressed in petals of all three-sex types and in stamens of hermaphrodite and male flowers. CpTM6-1 expressed at low levels in sepals and carpels, whereas CpTM6-2 expressed at a low level in sepals and at a high level in leaves. Our results showed that B-class gene homologs could function as predicted by the ABC model in trioecous flowers but differential expressions of CpTM6-1, and CpTM6-2, and CpPI suggested the diversification of their functions after the duplication events. Christine M. Ackerman, Qingyi Yu contributed equally to this work.     

Molecular variation of the testes-specific β<Emphasis Type="Italic">NACtes</Emphasis> genes in the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> genome

The βNACtes gene family of the Drosophila melanogaster genome provides a model for investigating the mechanisms of the molecular evolution of recently evolved genes. The βNACtes genes code for proteins that are homologous to the subunit of the nascent polypeptide-associated complex (NAC), are expressed exclusively in the testis, and are localized on the X chromosome as two-gene clusters and one separate copy. Population polymorphism of the βNACtes genes was studied using several wild-type D. melanogaster stocks, and βNACtes paralogs were compared with each other. A heterogeneous pattern was observed for βNACtes polymorphism: the 3′ genes of the two-gene clusters were low polymorphic, whereas, separate, the βNACtes1 gene was the most variable. The 5′ βNACtes copies of the two-gene tandems were practically identical, whereas the 3′ βNACtes copies were highly diverged. Hence, local gene conversion was assumed to provide for the selective homogenization of the 5′ genes. A comparison of the βNACtes paralogs showed that the majority of amino acid differences were in the N-terminal region, containing the βNAC domain. The McDonald-Kreitman test was used to analyze the divergence of βNACtes paralogs and implicated positive selection in the evolution of the βNACtes gene family.     

Specific Duplication and Dorsoventrally Asymmetric Expression Patterns of Cycloidea-Like Genes in Zygomorphic Species of Ranunculaceae

Floral bilateral symmetry (zygomorphy) has evolved several times independently in angiosperms from radially symmetrical (actinomorphic) ancestral states. Homologs of the Antirrhinum majus Cycloidea gene (Cyc) have been shown to control floral symmetry in diverse groups in core eudicots. In the basal eudicot family Ranunculaceae, there is a single evolutionary transition from actinomorphy to zygomorphy in the stem lineage of the tribe Delphinieae. We characterized Cyc homologs in 18 genera of Ranunculaceae, including the four genera of Delphinieae, in a sampling that represents the floral morphological diversity of this tribe, and reconstructed the evolutionary history of this gene family in Ranunculaceae. Within each of the two RanaCyL (Ranunculaceae Cycloidea-like) lineages previously identified, an additional duplication possibly predating the emergence of the Delphinieae was found, resulting in up to four gene copies in zygomorphic species. Expression analyses indicate that the RanaCyL paralogs are expressed early in floral buds and that the duration of their expression varies between species and paralog class. At most one RanaCyL paralog was expressed during the late stages of floral development in the actinomorphic species studied whereas all paralogs from the zygomorphic species were expressed, composing a species-specific identity code for perianth organs. The contrasted asymmetric patterns of expression observed in the two zygomorphic species is discussed in relation to their distinct perianth architecture.     

Phylogenetic Timing of the Fish-Specific Genome Duplication Correlates with the Diversification of Teleost Fish

For many genes, ray-finned fish (Actinopterygii) have two paralogous copies, where only one ortholog is present in tetrapods. The discovery of an additional, almost-complete set of Hox clusters in teleosts (zebrafish, pufferfish, medaka, and cichlid) but not in basal actinopterygian lineages (Polypterus) led to the formulation of the fish-specific genome duplication hypothesis. The phylogenetic timing of this genome duplication during the evolution of ray-finned fish is unknown, since only a few species of basal fish lineages have been investigated so far. In this study, three nuclear genes (fzd8, sox11, tyrosinase) were sequenced from sturgeons (Acipenseriformes), gars (Semionotiformes), bony tongues (Osteoglossomorpha), and a tenpounder (Elopomorpha). For these three genes, two copies have been described previously teleosts (e.g., zebrafish, pufferfish), but only one orthologous copy is found in tetrapods. Individual gene trees for these three genes and a concatenated dataset support the hypothesis that the fish-specific genome duplication event took place after the split of the Acipenseriformes and the Semionotiformes from the lineage leading to teleost fish but before the divergence of Osteoglossiformes. If these three genes were duplicated during the proposed fish-specific genome duplication event, then this event separates the species-poor early-branching lineages from the species-rich teleost lineage. The additional number of genes resulting from this event might have facilitated the evolutionary radiation and the phenotypic diversification of the teleost fish.[Reviewing Editor: Martin Kreitman]     

Selection on Length Mutations After Frameshift Can Explain the Origin and Retention of the <Emphasis Type="Italic">AP3/DEF</Emphasis>-Like Paralogues in <Emphasis Type="Italic">Impatiens</Emphasis>

Evolution of class B genes through gene duplication has been proposed as an evolutionary mechanism that contributed to the enormous floral diversity. Frameshift mutations are a likely mechanism to explain the divergent C-terminal sequences of MIKC gene subfamilies. So far, the inferences for frameshifts and selective pressures on the C-terminal domain are made for old duplications for which the exact selective pressures are obscured by evolutionary time. This motivated us to study an example of a recent duplication, which allows us to consider in more detail the selective pressures that are involved after duplication. We find that after duplication and frameshift of Impatiens class B genes, the individual codons show no evidence for adaptive selection. It is rather the length of the C-terminal domain that either is strictly conserved or varies strongly. This suggests a role for the length of the C-terminal domain in the retention of duplicated genes.     

Evolution of the APETALA3 and PISTILLATA lineages of MADS-box-containing genes in the basal angiosperms

The B class genes, including homologs of the Arabidopsis loci APETALA3 (AP3) and PISTILLATA (PI ), appear to play a conserved role in the determination of petal and stamen identity across core eudicot angiosperms. Understanding how and when these functions evolved is a critical component of elucidating the evolution of flowers, particularly the appearance of petaloid perianth organs. Before comparisons of gene expression patterns or functions can be made, however, it is necessary to establish the orthology of AP3 and PI homologs from basal angiosperms. Here, we report the identification and analysis of 29 new representatives of the B gene lineage from basal ANITA and magnoliid dicot angiosperms. These studies indicate that gene duplications have occurred at every phylogenetic level, both before and after the duplication that produced the separate AP3 and PI lineages. Comparison of genomic structure among PI homologs indicates that a 12-nucleotide deletion that had been considered synapomorphic for the whole PI lineage actually arose within the ANITA grade, after the split of the Nymphaeales but before the separation of the Austrobaileyales. Evidence for alternative splicing of the Nymphaea AP3 homolog is also presented. The implications of these findings for angiosperm systematics, the conservation of AP3 and PI gene function, and the evolution of the ABC program are discussed.     

Characterization of candidate class A,B and E floral homeotic genes from the perianthless basal angiosperm <Emphasis Type="Italic">Chloranthus spicatus</Emphasis> (Chloranthaceae)

The classic ABC model explains the activities of each class of floral homeotic genes in specifying the identity of floral organs. Thus, changes in these genes may underlay the origin of floral diversity during evolution. In this study, three MADS-box genes were isolated from the perianthless basal angiosperm Chloranthus spicatus. Sequence and phylogenetic analyses revealed that they are AP1-like, AP3-like and SEP3-like genes, and hence these genes were termed CsAP1, CsAP3 and CsSEP3, respectively. Due to these assignments, they represent candidate class A, class B and class E genes, respectively. Expression patterns suggest that the CsAP1, CsAP3 and CsSEP3 genes function during flower development of C. spicatus. CsAP1 is expressed broadly in the flower, which may reflect the ancestral function of SQUA-like genes in the specification of inflorescence and floral meristems rather than in patterning of the flower. CsAP3 is exclusively expressed in male floral organs, providing the evidence that AP3-like genes have ancestral function in differentiation between male and female reproductive organs. CsSEP3 expression is not detectable in spike meristems, but its mRNA accumulates throughout the flower, supporting the view that SEP-like genes have conserved expression pattern and function throughout angiosperm. Studies of synonymous vs nonsynonymous nucleotide substitutions indicate that these genes have not evolved under changes in evolutionary forces. All the data above suggest that the genes may have maintained at least some ancestral functions despite the lack of perianth in the flowers of C. spicatus. Nucleotide sequences data from this article have been deposited with the EMBL/GenBank Data Libraries under accession numbers AY316311, AY397762 and AY379963.     

三叶木通花粉前纤维蛋白基因的克隆与表达

以三叶木通花蕾为材料,采用RT-PCR、3-′RACE方法克隆了三叶木通花粉前纤维蛋白基因,命名为Atf-Pro(GenBank登录号GQ478584)。结果表明:AtfPro的cDNA全长735 bp、阅读框393 bp、编码131个氨基酸,有1个342 bp的3′端非翻译区。预测分子量约为14.081 kD,等电点4.74。氨基酸和核苷酸序列的同源性分析发现,AtfPro基因属于植物花粉profilin基因家族的新成员。RT-PCR定性分析表明,AtfPro基因在三叶木通花蕾、花药、雌花花瓣和柱头组织中均有表达,但在幼叶、茎尖、根尖组织中低水平表达或不表达,生殖器官中的表达时期从花序分化发育开始到开花散粉结束。     

Selection for Antimicrobial Peptide Diversity in Frogs Leads to Gene Duplication and Low Allelic Variation

Antimicrobial peptides are highly diverse pathogen-killing molecules. In many taxa, their evolution is characterized by positive selection and frequent gene duplication. It has been proposed that genes encoding antimicrobial peptides might be subject to balancing selection and/or an enhanced mutation rate, but these hypotheses have not been well evaluated because allelic variation has rarely been studied at antimicrobial peptide loci. We present an evolutionary analysis of novel antimicrobial peptide genes from leopard frogs, Rana. Our results demonstrate that a single genome contains multiple homologous copies, among which there is an excess of nonsynonymous nucleotide site divergence relative to that expected from synonymous site divergence. Thus, we confirm the trends of recurrent duplication and positive selection. Allelic variation is quite low relative to interspecies divergence, indicating a recent positive selective sweep with no evidence of balancing selection. Repeated gene duplication, rather than a balanced maintenance of divergent allelic variants at individual loci, appears to be how frogs have responded to selection for a diverse suite of antimicrobial peptides. Our data also support a pattern of enhanced synonymous site substitution in the mature peptide region of the gene, but we cannot conclude that this is due to an elevated mutation rate.     

<Emphasis Type="Italic">APETALA2</Emphasis> like genes from <Emphasis Type="Italic">Picea abies</Emphasis> show functional similarities to their Arabidopsis homologues

In angiosperm flower development the identity of the floral organs is determined by the A, B and C factors. Here we present the characterisation of three homologues of the A class gene APETALA2 (AP2) from the conifer Picea abies (Norway spruce), Picea abies APETALA2 LIKE1 (PaAP2L1), PaAP2L2 and PaAP2L3. Similar to AP2 these genes contain sequence motifs complementary to miRNA172 that has been shown to regulate AP2 in Arabidopsis. The genes display distinct expression patterns during plant development; in the female-cone bud PaAP2L1 and PaAP2L3 are expressed in the seed-bearing ovuliferous scale in a pattern complementary to each other, and overlapping with the expression of the C class-related gene DAL2. To study the function of PaAP2L1 and PaAP2L2 the genes were expressed in Arabidopsis. The transgenic PaAP2L2 plants were stunted and flowered later than control plants. Flowers were indeterminate and produced an excess of floral organs most severely in the two inner whorls, associated with an ectopic expression of the meristem-regulating gene WUSCHEL. No homeotic changes in floral-organ identities occurred, but in the ap2-1 mutant background PaAP2L2 was able to promote petal identity, indicating that the spruce AP2 gene has the capacity to substitute for an A class gene in Arabidopsis. In spite of the long evolutionary distance between angiosperms and gymnosperms and the fact that gymnosperms lack structures homologous to sepals and petals our data supports a functional conservation of AP2 genes among the seed plants.     

Identification of novel oocyte and granulosa cell markers

Here we present novel gene expression patterns in the ovary as part of an ongoing assessment of published micro-array data from mouse oocytes and embryos. We present the expression patterns of 13 genes that had been determined by micro-array to be expressed in the mature egg, but not during subsequent preimplantation development. In-situ hybridization of sectioned ovaries revealed that these genes were expressed in one of two distinct patterns: (1) oocyte-specific or (2) expressed in both the oocyte and surrounding granulosa cells. Despite the fact that micro-array data demonstrated expression in the egg, several of these genes are expressed at low levels in the oocyte, but strongly expressed in granulosa cells. Eleven of these genes have no reported function or expression during oogenesis, indicating that this approach is a necessary step towards functional annotation of the genome. Also of note is that while some of these gene products have been well characterized in other tissues and cell types, others are relatively unstudied in the literature. Our results provide novel gene expression information that may provide insights into the molecular mechanisms of follicular recruitment, oocyte maturation and ovulation and will direct further experimentation into the role these genes play during oogenesis.     

Heterotopic expression of class B floral homeotic genes supports a modified ABC model for tulip (Tulipa gesneriana)

In higher eudicotyledonous angiosperms the floral organs are typically arranged in four different whorls, containing sepals, petals, stamens and carpels. According to the ABC model, the identity of these organs is specified by floral homeotic genes of class A, A+B, B+C and C, respectively. In contrast to the sepal and petal whorls of eudicots, the perianths of many plants from the Liliaceae family have two outer whorls of almost identical petaloid organs, called tepals. To explain the Liliaceae flower morphology, van Tunen et al. (1993) proposed a modified ABC model, exemplified with tulip. According to this model, class B genes are not only expressed in whorls 2 and 3, but also in whorl 1. Thus the organs of both whorls 1 and 2 express class A plus class B genes and, therefore, get the same petaloid identity. To test this modified ABC model we have cloned and characterized putative class B genes from tulip. Two DEF- and one GLO-like gene were identified, named TGDEFA, TGDEFB and TGGLO. Northern hybridization analysis showed that all of these genes are expressed in whorls 1, 2 and 3 (outer and inner tepals and stamens), thus corroborating the modified ABC model. In addition, these experiments demonstrated that TGGLO is also weakly expressed in carpels, leaves, stems and bracts. Gel retardation assays revealed that TGGLO alone binds to DNA as a homodimer. In contrast, TGDEFA and TGDEFB cannot homodimerize, but make heterodimers with PI. Homodimerization of GLO-like protein has also been reported for lily, suggesting that this phenomenon is conserved within Liliaceae plants or even monocot species.these authors contributed equally to this work     

Functional analysis of gene duplications in Saccharomyces cerevisiae

Gene duplication can occur on two scales: whole-genome duplications (WGD) and smaller-scale duplications (SSD) involving individual genes or genomic segments. Duplication may result in functionally redundant genes or diverge in function through neofunctionalization or subfunctionalization. The effect of duplication scale on functional evolution has not yet been explored, probably due to the lack of global knowledge of protein function and different times of duplication events. To address this question, we used integrated Bayesian analysis of diverse functional genomic data to accurately evaluate the extent of functional similarity and divergence between paralogs on a global scale. We found that paralogs resulting from the whole-genome duplication are more likely to share interaction partners and biological functions than smaller-scale duplicates, independent of sequence similarity. In addition, WGD paralogs show lower frequency of essential genes and higher synthetic lethality rate, but instead diverge more in expression pattern and upstream regulatory region. Thus, our analysis demonstrates that WGD paralogs generally have similar compensatory functions but diverging expression patterns, suggesting a potential of distinct evolutionary scenarios for paralogs that arose through different duplication mechanisms. Furthermore, by identifying these functional disparities between the two types of duplicates, we reconcile previous disputes on the relationship between sequence divergence and expression divergence or essentiality.